1. Field of the Invention
This invention generally relates to a carboxypeptidase R inhibitor. More particularly, this invention relates to a peptide which inhibits activity of carboxypeptidase R so as to be effectively used in treatment of cerebral infarction, myocardial infarction and disseminated intravascular coagulation (DIC)
2. Background Art
Carboxypeptidases (CPs) are enzymes which catalyze the hydrolysis of peptide bonds at the C-terminus of peptides and proteins. They have been categorized according to their actions, either as metallocarboxypeptidases (MPs) or cysteine/serine-carboxypeptidases (Cys/Ser-CP). MPs are distinct from Cys/Ser-CPs. MPs possess a tightly bound Zn2+ atom, which is directly involved in catalysis, while Cys/Ser-CPs contain a reactive Cys/Ser residue at their active site as the Ser/His/Asp triad of serine proteases. MPs can be further subdivided into carboxypeptidase A-like enzymes having a preference for hydrophobic C-terminal residues, and carboxypeptidase B-like enzymes having a preference for C-terminal basic residues (lysine and arginine). For review see Vendrell et al. [1].
Carboxypeptidase R (CPR) [2], also known as plasma carboxypeptidase B (CPB) [3], carboxypeptidase U (CPU) [4] or activated thrombin-activatable fibrinolysis inhibitor (TAFIa) [5], is a carboxypeptidase B-like enzyme present as a zymogen (proCPR) in plasma. CPR plays a crucial role in regulating fibrinolysis [3, 6] and acts as an inactivator of inflammatory mediators, preferentially removing C-terminal arginine (R) residues from anaphylatoxins, thereby preventing excess inflammation [7]. Carboxypeptidase N (CPN; 270 kDa glycoprotein) [8] which is also present in plasma is also an important inactivator of anaphylatoxins, kinins and fibrinopeptides [9–11]. However, in contrast to CPR, it does not play any significant role in dampening fibrinolysis. Moreover, CPN is present in an active form in plasma whereas CPR is found in the inactive proCPR state and is called into play during coagulation [2]. Previously, the carboxypeptidase activity in plasma which is responsible for inactivation of bradykinin, anaphylatoxins and other basic carboxy-terminal peptides was considered due to CPN alone [9–11]. However, CPR was also shown to inactivate bradykinin [12, 13] and furthermore, CPR, not CPN, was found to effectively reduce fibrinolysis [14].
Following its activation, CPR catalyses the removal of C-terminal lysine (K) residues from cell-surface proteins and partially degraded fibrin clots thereby preventing the binding [15, 16] and activation of plasminogen [5, 14]. Conversion of plasminogen (Glu-plasminogen) to its active form, plasmin (Lys-plasmin or Lys-plasminogen), requires tissue-plasminogen activator (t-PA) which is an activator of blood clot lysis. Binding of t-PA to fibrin via its K-binding domain catalyses cleavage of Glu-plasminogen (i.e. plasminogen) at the C-terminus of Lys76 to yield Lys-plasminogen (i.e. plasmin). Lys-plasminogen has an increased affinity for fibrin compared with Glu-plasminogen since the release of residues 1–76 exposes the kringle 1 domain, which contains two residues (Arg32 and Arg34), which were considered to be responsible for fibrin binding [17]. Studies have shown that CPR inhibits t-PA-induced lysis only when Glu-plasminogen, not Lys-plasminogen, is present, suggesting that its antifibrinolytic effect is primarily mediated through inhibition of Glu-plasminogen activation [5]. Therefore, removal of K-residues by CPR from partially degraded fibrin diminishes the already low binding affinity of Glu-plasminogen to fibrin, essentially prolonging fibrinolysis [3]. Plasmin-mediated proteolysis of fibrin constitutes a positive-feedback process that enhances plasminogen activation. CPR inhibits fibrinolysis by removing C-terminal lysine residues from fibrin, thereby limiting plasmin formation [5, 13, 14].
At present, t-PA is the only treatment for thromboembolic stroke approved by the Food and Drug Administration. However, recently adverse side effects have been reported in some patients, suggesting that t-PA may modulate N-methyl-D-aspartate (NMDA)-receptor-mediated signaling and excitotoxic neuronal death [18]. To alleviate this problem, the co-administration of potato carboxypeptidase inhibitor (PCI) with lower doses of t-PA has been suggested and recent experiments in animal models have shown that PCI dramatically enhances clot lysis [19]. PCI [20] is a small 39-residue protein (MW 4295 Da) which has the ability to selectively inhibit CPR without affecting the activity of CPN in the circulation [14]. An understanding of the interaction between CPR and PCI is crucial for the development of novel therapeutics for use in thrombolytic therapy. The inventors' studies have lead to the design of a smaller peptide inhibitor of CPR, designated as CPI-2KR, which shows similar biological activity to PCI in vitro and is a competitive inhibitor of CPR. While this peptide may be useful for the treatment and/or prevention of thrombosis, it also represents a starting point for the design of low molecular weight organic molecules, which are preferable and more useful alternatives. In this description, the rational structure-based strategies employed to design the novel CPR inhibitor are also discussed.